Food scientists have long used hyperbaric conditions to reduce the microbial burden of foodstuffs. Vaccine biologists are also very interested in inactivating microorganisms, for their use in vaccine preparations. However, to make a safe, effective vaccine, one must 1) completely inactivate the pathogenic microorganism; and 2) retain the organism's immunogenic potential (immunogenicity). Before the instant invention was made, the skilled person knew certain combinations of pressure, temperature, and time could be used to reduce the number of viable microorganisms, however, he or she did not have a generally-applicable method to produce “vaccine-suitable” inactivated microorganisms. For examples of hyperbaric reduction of biological load in food, see e.g. Isbarn, 2007 (influenza); Ritz, 2000 (salmonella); and Wilkinson, 2001 (poliovirus). Up until recently, any retention of immunogenic potential by the microorganisms was merely incidental to the goal of reducing microbial burden.
As regards use of high pressure inactivation for vaccine production, one group achieved good inactivation of Leptospira interrogans serovar hardjo by subjecting the microorganisms to two kilobar for sixty minutes (Silva, 2001). The inactivated leptospires were able to elicit immune responses in rabbit, though their ability to elicit protective immune response in a target animal, such as a bovine, was not demonstrated. To date, Applicants are aware of no published results demonstrating complete protective immunity using pressure-inactivated bacteria or protozoal parasites. For a review, please see Shearer et al., 2009. In addition to not yet providing effective vaccines using hyperbaric inactivation methods, the field has yet to produce the necessary hyperbaric devices. Current high hydrostatic pressure (HHP) devices developed for merely reducing microbial burden lack the ability to completely inactivate pathogens and render them useful as vaccine constituents.
Moreover, existing hyperbaric methods introduce unacceptable, heterogeneous temperature distributions, which result in reduced yields for protein folding/solubilization and pathogen inactivation applications. Available devices affect pressure increases by using external pumps to inject additional liquid into a fixed-size vessel. Essentially, pumps outside the vessel pressurize the water, and as more water is injected into the vessel, the pressure increases, and an arrangement of valves maintains the desired pressure within the chamber. The temperature distribution problem stems from the pump because as one increases the pressure inside the pump, the water becomes very hot. Injecting the high pressure, hot water produces the high degree of temperature heterogeneity. These devices thus may be perfectly adequate for reducing pathogen burden in foodstuffs, where precision regulation of temperature and pressure is not required, but these devices cannot be efficiently applied to refold/solubilize protein or inactivate pathogens while maintaining their immunogenic potential. Companies working in this area include Barofold (see for example, U.S. Pat. Nos. 6,489,450, 7,064,192, 7,538,198, 7,767,795, 7,829,681, 8,329,878, and US20080161242A1) and Avure, which makes hyperbaric devices for the food processing industry.
As the prior art devices do not allow for optimal control over temperature and pressure, hyperbaric devices designed to provide precise control over these variables are required to serve facilitate immunogenicity-preserving pathogen inactivation and protein refolding/solubilization/solubilization.
Applicants have therefore developed specific hyperbaric methods and devices to produce vaccine-ready inactivated microorganisms and to refold/solubilize commercially relevant therapeutic and immunogenic proteins.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.